title: Enhanced transgene expression in sugarcane by co-expression of virus-encoded RNA silencing suppressors.
abstract: Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.
authors.affiliation: Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China.
authors.forename: San-Ji
authors.initials: SJ
authors.lastname: Gao
authors.forename: Mona B
authors.initials: MB
authors.lastname: Damaj
authors.forename: Jong-Won
authors.initials: JW
authors.lastname: Park
authors.forename: Getu
authors.initials: G
authors.lastname: Beyene
authors.forename: Marco T
authors.initials: MT
authors.lastname: Buenrostro-Nava
authors.forename: Joe
authors.initials: J
authors.lastname: Molina
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.forename: Jessica J
authors.initials: JJ
authors.lastname: Ciomperlik
authors.forename: Shuga A
authors.initials: SA
authors.lastname: Manabayeva
authors.forename: Veria Y
authors.initials: VY
authors.lastname: Alvarado
authors.forename: Keerti S
authors.initials: KS
authors.lastname: Rathore
authors.forename: Herman B
authors.initials: HB
authors.lastname: Scholthof
authors.forename: T Erik
authors.initials: TE
authors.lastname: Mirkov
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D018628:Gene Dosage; D015870:Gene Expression; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D016153:Genes, Suppressor; D005814:Genes, Viral; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D009029:Mosaic Viruses / Q000235:genetics*; D019697:Onions; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000821:virology; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics; D030821:Plants, Genetically Modified; D034622:RNA Interference; D031786:Saccharum / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D014026:Nicotiana; D017864:Tombusvirus / Q000235:genetics*; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D010940:Plant Proteins; C054562:yellow fluorescent protein, Bacteria; D005966:Glucuronidase